Method for the selective degradation of milk protein in the presence of other milk proteins

ABSTRACT

A method is presented for producing a casein/caseinate hydrolysate stripped of immunogenic proteins. The method comprises treating an aqueous solution, such as milk, comprising casein/caseinate and immunogenic protein other than casein with a protease that selectively hydrolyzes casein. After the casein/caseinate has been hydrolyzed, the casein/caseinate hydrolyzate is separated by membrane ultrafiltration from unhydrolyzed immunogenic protein and recovered. The resulting hydrolyzate is essentially free of antigenic components of the ABBOS peptide and bovine serum albumin. The casein selective protease is selected from cell wall proteinase of  Lactococcus lactis  subsp. Cremoris Wg2, a  Clostridium histolyticum  collagenase and a  Bacillus polymyxa  dispase.

The present invention relates to a method for the selective degradationof milk proteins, and in particular to a method for the selectivehydrolysis of casein and/or casein/caseinate in the presence of at leastone further protein constituent other than casein/caseinate.

The invention more in particular relates to such a method wherein thefurther protein constituent is a milk protein (constituent) other thancasein, in particular a whey protein (constituent), and/or to such amethod wherein the casein/caseinate and preferably also the at least onefurther protein constituent are essentially in solution, in particularessentially in an aqueous solution.

According to the method of the invention, the casein/caseinate isspecifically degraded in the presence of the at least one furtherprotein constituent, said further protein constituent essentiallyremaining intact in the process. This allows the casein hydrolysisfragments to be separated, in a further separation step, from the one ormore further protein constituents which have essentially remainedintact.

The invention is based on the surprising finding that certainproteinases exhibit a very high specificity with respect to casein inthe presence of other proteins, in particular in the presence of othermilk proteins such as whey proteins. The invention is further based onthe use of these specific proteinases in the hydrolysis ofcasein-containing protein preparations, in particular caseinpreparations or (aqueous) solutions of milk proteins, such as milk.

The method of the invention can be used, in a first embodiment, for thepreparation of a casein hydrolysate which has been stripped of one ormore immunogenic protein components other than casein. In a secondembodiment, the invention can be used for preparing a milk proteinpreparation, in particular a whey protein preparation which isessentially stripped of casein/caseinate, starting from acasein-containing milk protein preparation such as milk, whey or asolution of milk or whey proteins. These whey protein preparationsstripped of casein likewise have beneficial properties from animmunological point of view.

The invention will be further discussed with reference to thesespecific, non-limiting embodiments “A” and “B”, which are schematicallyshown in FIG. 6. Both embodiments comprise the step of selectivelydegrading casein in the presence of at least one further proteinaceouscomponent, and separating the hydrolysed fragments from the non-degradedproteins, but differ in the starting material used, the product desired,as well as the intended use and required (immunological) properties ofsaid product, as will become clear hereinbelow.

EP-A-0 250 501 describes a method for producing a whey proteinhydrolysate, comprising the steps of:

a) removing casein from the whey protein starting material;

b) hydrolysis of the casein-free protein material obtained in (a) withat least one protease;

c) ultrafiltration of the hydrolysate of (b) using a membrane with a“cut-off” value of no more than 20,000 Dalton;

and optionally further processing of the filtate thus obtained.

In step a), the casein is removed from the whey protein startingmaterial by either physical separation such as precipitation orfiltration; or enzymatically by the use of the neutral metalloproteaseof Bacillus subtilis (Neutrase®, Novo Industrie A/S), which is said tohave “a high activity against casein but no or only a weak activityagainst both native as well as heat-denatured whey proteins”.

The enzymatic hydrolysis is carried out at a pH of 4-9, preferably pH7.5, and a temperature of 30-60° C., preferably 50° C., until“completion”, which is said to be a degree of hydrolysis (DH) of about4%, based upon total protein. The casein fragments are then removed bymeans of centrifugation or ultrafiltration using—when the whey proteinstarting material has not been heat-treated—a membrane with a “cut-off”value of no more than 20,000.

Because the molecular weight of the different caseins (19.5-25 kDa) arein the same range as the molecular weights of the native whey proteinsα-lactalbumin (14.2 kDa) and β-lactoglobulin (18.2 kDa (monomer)) it isdoubtful whether the use of a 20 kDa membrane will lead to theseparation of the hydrolysed caseins from the whey proteins.

The “casein-free” whey proteins thus obtained are then furtherhydrolysed using enzymes such as Alkalase® 2.4 L and Rhozyme P41®, andthen ultrafiltrated. According to this disclosure, by selectivelyremoving the casein before the whey proteins are hydrolysed, the finalwhey protein hydrolysate will have a less bitter taste.

However, the casein depleted whey protein preparation obtained aftersaid step a) is only an intermediate product, and not the end product.Accordingly, no immunological determination of the allergenic propertiesof this intermediate product is given. In the method of EP-A-0 250 501,this is also not essential, as the proteins obtained are hydrolysedfurther, after which any still remaining antigenic fragments can beremoved by ultrafiltration. However, from the low degree of hydrolysis(4%) in step a), it is expected that at least some antigenic caseinfragments will be present in said intermediate product.

EP-A-0 384 303 describes a method for hydrolysing proteins, especiallymilk proteins and whey proteins, in which a combination of a proteinasepreparation from Aspergillus sp. and a bacterial aminopeptidase is used.The use of this combination provides an end product with a less bittertaste, at a higher degree of hydrolysis: with said combination, the“bitter point” is reached at a DH of 4.4%, whereas the bitter point issaid to be reached at 1.2% when only the proteinase is used.

EP-A-0 610 411 discloses a method for obtaining a casein hydrolysate, inwhich a casein or caseinate is suspended in an aqueous medium andhydrolyzised to a DH of 15-35%, preferably 22-28%, using a combinationof proteases from the following three groups:

1) one or more neutral endoproteases of a Bacillus (such as Neutrase®);

2) one or more basic endoproteases of Bacillus (such as Alcalase®,Esperase® and Savinase®) and

3) an endoprotease of Aspergillus (such as Novozym® 515).

According to this reference, the use of such a combination of proteases,and hydrolysis to a DH of 15-35% (compared to 4.4% such as disclosed inEP-A-0 250 501 as well as for instance EP-A-0 384 303) provides animproved casein hydrolysate. Nevertheless, no immunologicaldetermination of the antigenic properties of the hydrolysate thusobtained is disclosed. Also, the enzymes used are obtained from Bacillussubtilis, which is not a food grade microorganism.

EP-A-0 631 731 describes a method for producing a partial hydrolysate ofmilk protein by enzymatic hydrolysis of a mixture of whey protein andcasein to a DH between 4 and 10%. Although a reduction in antigenicityof 80% or more was obtained, according to the examples the resultinghydrolysates still had some residual antigenicity as determined by anELISA.

EP-A-0 421 309 describes a method for preparing a whey proteinhydrolysate free of allergenics by pepsin prehydrolysis, followed bytrypsin-chymotwpsin hydrolysis in the presence of a cathionic serineendoprotease of type elastase 2.

Derwent Abstracts AN-96-471202 and AN-94-337356 (corresponding toJP-08238059-A and JP-06261691-A) disclose a method for producing alow-allergenic milk protein preparation, in which alpha-casein isselectively decomposed using a fungus derived protease, in particularfrom the geni Mucor and Cladosporium.

Derwent Abstract AN-95-307065 (corresponding to JP027203844 A) disclosesa method for preparing an emulsified whey protein hydrolysate with goodthermo-stability and up to 0.0001 residual antigen in ELISA, obtained byhydrolysing a solution of milk whey protein with a mixture of endotypeproteases from Bacillus subtilis, trypsin and papain.

EP-A-0 601 802 describes a method for removing allergenic compounds fromproteinaceous compositions, in which the protein in said composition isdecomposed with proteolytic enzymes into a protein hydrolysate having adegree of hydrolysis of 20-60%, followed by clarification of thehydrolysate thus obtained, preferably by centrifugation orultrafiltration.

Macropeptides still remaining in the intermediate product thus obtained(which according to Table 2 still contains β-lactoglobuline as shown onELISA) are then removed using an absorption resin. As the proteolyticenzyme trypsin, pancreatin or microbial proteases such as Alkalase®0.6 Lfrom Bacillus licheniformis can be used.

B. Kiefer-Partsch et al., Appl.Microbiol.Biotechnol. (1989) 31:75-78describes the purification of an X-prolyl-dipeptidyl aminopeptidase fromthe cell wall proteolytic system of Lactococcus lactis sp. cremoris.This enzyme is described as a metal-independent, highly β-caseinspecific protease; reference is made to similar enzymes from L. lactisen Streptococcus thermophillus. These enzymes are however not used forproducing a casein hydrolysate, or specifically removing casein from awhey protein mixture.

A. Preparafion of a Casein Hydrolysate which Has Been Stripped of One orMore Protein Constituents Other than Casein

This embodiment of the invention relates to the preparation of a caseinhydrolysate which has been stripped of one or more immunogenic proteincomponents other than casein.

This embodiment particularly relates to a method for preparing a caseinhydrolysate free from ABBOS epitopes, and to the casein hydrolysate freefrom ABBOS epitopes thus obtained.

An ABBOS epitope is to be understood, hereinafter, as that part of theantigenic ABBOS peptide/fragment of bovine serum albumin (BSA) whichreacts with an antibody directed to the ABBOS peptide.

A casein hydrolysate free from ABBOS epitopes is to be understoodhereinafter as a casein/caseinate hydrolysate which does not contain anypeptides or peptide fragments which may form a bond with antibodies toABBOS epitope (cross-reaction).

It is known that proteins from cow's milk may give rise, because oftheir antigenic properties, to immunological reactions, particularly inneonates/infants. These immunological reactions can manifest themselves,for example, as hypersensitivity reactions (cow's milk protein allergy).

One of the immunological reactions against proteins from cow's milk,which has received a great deal of attention in recent years, is thereaction against (epitopes of) bovine serum albumin (BSA) in infants,because of the role played by this immune response in the onset of typeI Diabetes Mellitus.

There are indications that, in infants, immunoglobulin G (IgG)antibodies against a specific region of BSA (amino acids 126-144, theso-called ABBOS peptide) can be generated which, under certainconditions, are cross-reactive with the homologous structure in the P69protein on the surface of pancreatic beta cells. This may give rise toan autoimmune reaction leading to the destruction of the beta cells andconsequently to a failure in insulin production.

These findings are based, inter alia, on the fact that the ABBOS epitopeis not completely broken down in the digestive tract, even though itcontains a large number of possible attack/cleavage sites for theproteolytic enzymes in the gastrointestinal tract. Earlier studies haveproduced, inter alia, the following results:

in the blood of infants in which diabetes has been diagnosed, antibodiesto BSA and in particular to the ABBOS peptide can be detected;

the physiological digestion of BSA proceeds less efficiently in infantsthan in adults, due to the higher pH in the stomach;

The result is that fragments of BSA containing the intact epitope of theABBOS peptide are still present after digestion. As in infants, someparts of the mucosal barrier are not yet completely developed and/orharmonized, these large peptide fragments can pass through theintestinal wall and reach the blood stream. These foreign proteins wouldthen stimulate the immune system of the infant to produce antibodieswhich may possibly be directed against the ABBOS epitope.

Other proteins in cow's milk which may give rise to immunologicalreactions are, inter alia, α-lactalbumin (α-Lac), beta-lactoglobulin(β-Lg), bovine immunoglobulin G (IgG) and the caseins, as well asantigenic fragments thereof.

In the prior art foods have therefore been developed, in particular forinfant formulas, which are based on cow's milk protein which has beendegraded/hydrolysed by certain enzymes, resulting in a reduction in theantigenic properties of the milk proteins. Milk protein hydrolysates(“hypoallergenic hydrolysates”) for the preparation of such foods arelikewise commercially available.

Such hydrolysates are obtained by the treatment of milk protein withproteolytic enzymes (proteinases), leading to the degradation of boththe casein and/or the caseinate, which—in terms of protein (determinedas dry substance)—forms the main constituent (>80 wt %) of milkproteins, as well as all the other proteins present, including theantigenic proteins and their epitopes.

Studies by the applicant have shown, however, that the commerciallyavailable milk protein hydrolysates generally still give cross-reactionsand/or exhibit antigenic properties, particularly in specific in vitroimmunological assays such as enzyme-linked immunosorbent assay (ELISA).For example, these known hydrolysates still contain BSA fragments whichexhibit cross-reactivity with the ABBOS peptide and/or contain intactABBOS epitopes. Studies by the applicant have demonstrated the presenceof these epitopes by means of immunological assay techniques usingABBOS-specific antibodies.

These cross-reactive fragments/immunogenic components could in theory beremoved by a higher degree of hydrolysis of the milk proteinhydrolysates. This, however, leads to an unduly high level of undesired“free” amino acids in the final product, which are less well absorbedthrough the intestinal wall and, moreover, may detract from theproperties of the product, in particular its consistency and osmolality.The known hydrolysis processes cannot be adequately controlled in thisrespect.

It is therefore a first object of the invention to provide a milkprotein preparation, in particular a casein/caseinate hydrolysate, whichhas improved immunological properties.

It is a further object of the invention to provide such a preparationwhich has an acceptably low level of free amino acids.

In particular it is an object of the invention to provide acasein/caseinate hydrolysate in which essentially no antigeniccomponents of the ABBOS peptide are still present/detectable.

Further objectives and advantages of the invention will become clearfrom the following description.

We have now found that certain proteinases from lactococci exhibit veryhigh specificity to casein/caseinate, i.e. that they degrade thecasein/caseinate in milk protein very selectively, whereas the otherprotein constituents present, with the antigenic epitopes, remainessentially intact.

This surprising finding makes it possible, after prior hydrolysis of themilk protein, for the (relatively) small casein hydrolysis products tobe separated, in a simple manner, from the still virtually intactantigenic (whey) proteins, giving a casein hydrolysate which hasessentially been stripped of one or more specific antigenic componentssuch as antigenic whey proteins, and in particular of antigeniccomponents of BSA such as the epitope of the ABBOS peptide.

The invention therefore relates, in a first aspect, to a method forpreparing a hydrolysed milk protein preparation, starting from a milkprotein preparation which comprises more than 80 wt %, preferably morethan 90 wt %, of casein/caseinate and at least one further (milk)protein constituent, in which the milk protein preparation is broughtinto contact with a proteolytic enzyme under conditions for thehydrolysis of the casein/caseinate, characterized in that thecasein/caseinate and preferably also the at least one proteinconstituent are in an essentially dissolved state, and in that theproteolytic enzyme is a proteinase specific for casein/caseinate, suchthat the casein/caseinate is hydrolysed while the at least one (milk)protein constituent remains essentially intact.

In a further aspect, the invention relates to a method for preparing amilk protein hydrolysate which has been stripped of at least oneantigenic protein constituent, starting from a milk protein preparationwhich comprises more than 80 wt %, preferably more than 90 wt %, ofcasein/caseinate and the at least one antigenic protein constituent,comprising the steps of:

a. bringing the milk protein preparation into contact with a proteinasespecific for casein/caseinate, the casein/caseinate and preferably alsothe at least one protein constituent being in an essentially dissolvedstate, such that the casein/caseinate is hydrolysed while the at leastone antigenic protein constituent remains essentially intact,

b. separating the casein hydrolysis fragments and the at least oneantigenic protein constituent which has remained essentially intact.

In particular, the invention relates to such a method for preparing ahydrolysed casein preparation which does not contain any (antigeniccomponents of) BSA, in particular no (epitopes of the) ABBOS peptide.

The invention further relates to milk protein hydrolysates obtainableaccording to the method of the invention, in particular to such ahydrolysate which is “free from ABBOS epitopes”.

In the description and the claims, protein contents are expressed on thebasis of total protein, i.e. in per cent by weight based on the totalprotein content, in particular determined as dry substance, in theabsence of a statement to the contrary.

According to the invention, the casein/caseinate is essentially insolution, preferably in aqueous solution. The at least one furtherprotein constituent, and preferably (virtually) all further proteinconstituents present, are likewise preferably essentially in solution,more preferably in aqueous solution. The term “essentially in solution”should be understood as customarily used in the dairy industry, i.e.that the constituents in question are virtually entirely dissolved orare virtually entirely in a dissolved state, for example as a suspensionof micelles.

This means, incidentally, that there is an upper limit to thecasein/caseinate concentration—based on total solution—of the startingmaterial, depending on the solubility of casein in the medium used. Ingeneral, the casein concentration will be less than 20 wt % and inparticular less than 15 wt %, based on the total composition.

A casein-specific proteinase is to be understood, in particular, as aproteinase which degrades casein selectively in the presence of one ormore of the whey proteins α-Lac, β-Lg, BSA and IgG. The selectivity ofsuch proteinases is preferably such that, given equal ratios ofenzyme/substrate an within a given time period, the proteinase degradesmore than 90% of the intact casein and less than 5% of each of the wheyproteins mentioned.

It should be understood, however, that the invention does not excludethe possibility that the antigenic proteins to be removed are cleaved toa lesser extent—albeit much less rapidly than the casein/caseinate—toproduce (large) protein fragments, as long as these antigenic proteinfragments can be separated from the casein fragments obtained afterhydrolysis, the separation taking place, in particular, on the basis ofmolecular size or molecular mass.

The proteinases (also referred to as proteases) used in the inventionpreferably are derived from lactococci, more preferably from “foodgrade” lactococci, or possibly other suitable “food grade”microorganisms. In lactococci the proteinases are present, inparticular, on the outside of the cell wall.

Some nonlimiting examples of suitable proteinases, which may or may notbe derived from food grade organisms, are the cell wall proteinase ofLactococcus lactis subsp. cremoris, strain Wg2, collagenase(particularly from Clostridium histolyticum) and Dispase, (particularlyfrom Bacillus polymyxa) and the like, as well as the variants thereofobtained by recombinant-DNA techniques, if these have the desiredsubstrate specificity.

An example of a commercially available enzyme suitable for use in theinvention is Neutrase® (Novo Industri), although this may possiblyprovide a product with a somewhat more bitter taste and/or may possiblybe less selective, compared to the enzymes mentioned above.

Those skilled in the art will be able, on the basis of the presentdisclosure and/or by means of simple tests for determining thespecificity/activity with respect to casein and the further proteinconstituents of milk protein, to find other suitable proteolyticenzymes, which may be native or recombinant.

While a number of these proteinases and their use in cheese—making havebeen described in the prior art, their surprising substrate specificitywith respect to casein has not previously been reported or utilized.

The proteinases can be used in any suitable form, including enzymepreparations or enzyme isolates—which can be obtained in a manner knownper se and/or are commercially available—as well as in immobilized form.Mixtures of more than one proteinase can likewise be used, as long asthe desired specificity with respect to casein/caseinate is retained.

Owing to their specificity, the proteinases will in particularcleave/hydrolyse the protein bonds in the casein; in the widest sense,however, the invention is not limited to a specific type of enzymaticreaction.

In a further aspect, the invention relates to the use of theabove-described specific proteinases in the conversion of a milk proteinpreparation which comprises more than 80 wt %, preferably more than 90wt %, of casein/caseinate and at least one further protein constituent,the casein/caseinate and preferably also the at least one proteinconstituent being in an essentially dissolved state, preferably inaqueous solution.

The milk protein preparations which are used in the invention, inparticular casein preparations or casein-enriched milk or milk proteinpreparations, are commercially available and/or can be obtained in amanner known per se, starting from (cows') milk or milk products. Theinvention is however not limited to milk or milk preparations derivedfrom a specific animal. For instance, besides cows' milk, milk fromgoat, sheep, or any other mammal, or mixtures thereof, can also be usedas the starting product.

In addition to casein/caseinate (>80 wt %, preferably >90 wt %), thesepreparations generally contain further milk proteins such as BSA, α-Lac,β-Lg and IgG, in amounts of less than 20 wt %, in particular less than10 wt %. However, it will be clear to those skilled in the art that,where appropriate, milk or other protein preparations having low(er)casein levels, for example containing as low as 40 wt % of casein, canalso be used, although this may in some applications lead to a loweryield of casein hydrolysate, based on the starting preparation, andconsequently a less efficient process.

For instance, the starting product can be a mixture of casein or acasein containing protein preparation, and non-milk proteins, such assoy protein or a soy protein preparation. It is known that soy proteincan be used in foods, including infant formulas, to replace animalderived proteins or protein products, in order to reduce the allergenicproperties of said food. In such foods, soy protein can also be combinedwith casein hydrolysates or other milk protein hydrolysates.

Such foods can also be prepared according to the invention, for instanceby mixing a casein hydrolysate obtained according to the invention withthe desired amount of soy protein and further constituents known per se.

Alternatively, such foods can be prepared by providing a mixture ofcasein or a casein containing preparation (such as milk or a milkprotein preparation) and the soy protein, and selectively hydrolysingthe casein in said mixture in situ using a selective proteinase asdescribed herein. If required, any large intact proteins, such as thosealso described herein, can then be selectively removed. It will be clearto the skilled person that, in this latter embodiment of the invention,the casein concentration in the starting mixture will generally be lessthan 80%.

Therefore, in a further embodiment, the invention relates to a method—asdescribed herein—for selectively hydrolysing casein in a mixturecontaining at least one non-milk protein or protein component, such assoy protein, to provide a protein preparation containing at least thenon-milk protein and the hydrolysed casein fragments.

Another embodiment of the invention in which the casein concentration inthe starting preparation can be less than 80% comprises the use of astarting material comprising a mixture of casein or a casein containingpreparation, and milk protien or a milk protein preparation, forinstance in an amount of 5-50 wt %, preferably 20-40 wt % of themixture. Again, in such a mixture, the casein can be hydrolysedselectively in situ using the method described herein, after whichoptionally undesired peptides can be removed.

The hydrolysis of the milk protein preparation is performed in a mannerknown per se, under conditions suitable for the desired enzymaticconversion, such as a temperature of 20-40° C., a time of 4-16 hours,and a pH of 6-8, and using known equipment and techniques. In theprocess, the use of the specific proteinases may additionally provideimproved control of the enzymatic conversion.

The hydrolysis is carried out to a suitable extent, i.e. to a degreewhich allows the hydrolysed fragments to be separated from thenondegraded protein constituents, which is dependant upon factors suchas the antigenic components to be removed, the separation method to beused (in particular the molecular cut-off value of the ultrafiltrationmembrane to be used), and further factors clear to those skilled in theart.

The degree of hydrolysis is preferably such that any subsequentultrafiltration step can be carried out in a technically/industriallyacceptable manner (with respect to filtration rate, yield and the like).

The hydrolysis is terminated before too high a level of “free” aminoacids is obtained. The level of free amino acids in the preparationobtained after hydrolysis will, as a rule, be less than 5 wt %.

The progress of the hydrolysis can be monitored, if required, by asuitable technique such as DH (degree of hydrolysis) assay by measuring,for example, the consumption of alkali, or with the aid of the OPA(=O-phthalic anhydride) method, alternatively with the aid of achromatographic method such as RP-HPCL (reverse-phase high performanceliquid chromatography) analysis.

After the hydrolysis, the hydrolysed casein fragments can be separatedfrom the (virtually) intact, nondegraded other proteins or large proteinfragments. Any suitable separation method can be used for this purpose,in particular a separation method on the basis of molecular mass and/ormolecular size, especially ultrafiltration. This process canadvantageously make use of ultrafiltration equipment generally known inthe dairy industry, often already present within the dairy industryitself.

In ultrafiltration, use is made of a membrane having a suitablemolecular cut-off value, preferably 30 kDa or less, more preferably 10kDa or less, preferably less than 1 kDa, depending on the size of theantigenic proteins/protein components to be removed.

To obtain a preparation free from ABBOS epitopes, generally a membranewill be used having a molecular cut-off value of 30-10 kDa , IgG(molecular mass 146,000) being removed at the same time. To remove otherproteins, in particular smaller immunogenic components such as α-Lac andβ-LG, it is possible to use membranes having a molecular cut-off valueup to 5 kDa or less.

As a rule, the immunological purity of the hydrolysate will increase thefurther the caseins are hydrolysed and/or the lower the molecularcut-off value of the membrane used. The ultrafiltration rate (thethroughput through the membrane), and also the yield, will likewisedepend on the combination of the degree of hydrolysis and theultrafiltration membrane used.

The immunological purity of the hydrolysate can be assayed by means of asuitable immunological assay, in particular using one or more antibodieswhich are specific for the antigenic proteins or fragments thereof,and/or the epitopes in question. Suitable techniques such as ELISA andsuitable specific antibodies will be known to those skilled in the artand/or can be obtained in a manner known per se.

According to the invention, a hydrolysate is regarded as free from anantigenic component if, using such an immunological assay and inparticular ELISA, it is no longer possible to observe/detect anyantigenic reaction.

Although the invention allows immunologically highly pure hydrolysatesto be obtained, which are essentially free from a more than one, or all,of the immunogenic components present in milk protein, such asBSA/ABBOS, α-Lac and β-Lg and/or IgG, and are even free from immunogeniccomponents/epitopes of the casein/caseinate itself, the method accordingto the invention is especially suitable and intended for preparing ahydrolysate which is “BSA-free” or “free from ABBOS epitopes”.

In the preparation of such a “BSA/ABBOS-free” hydrolysate it is notrequired given that BSA is a relatively large protein—to hydrolyse to ashigh a degree as required for the (simultaneous) removal of smallerantigenic proteins such as α-Lac and/or β-Lg. Also, ultrafiltrationmembranes having a higher molecular cut-off value can be used, as statedabove. This has the advantage that the available equipment is lessoccupied (shorter reaction time for the hydrolysis, better throughput inthe ultrafiltration), resulting in a higher production per unit time.

It is therefore not excluded that, in a hydrolysate “free from ABBOSepitopes” according to the invention, (components/epitopes of) otherantigenic proteins, in particular smaller one, such as α-Lac and β-Lg,may still be detectable. Likewise it is possible for the preparations ofthe invention still to contain antigenic components of thecasein/caseinate itself.

The casein hydrolysates according to the invention can be further usedand/or processed in a manner known per se, for example for thepreparation of hypoallergenic foods such as infant formulas and enteralfoods, which can be used for preventing and/or treating cow's milkprotein allergy, in particular Diabetes Mellitus type I caused byimmunological reactions against BSA/ABBOS.

B. Preparing a Whey Protein Preparation Stripped of Casein/Caseinate

The second embodiment of the invention relates to the preparation of amilk protein preparation, in particular a whey protein preparation,which is essentially stripped of casein/caseinate, starting from acasein-containing milk protein preparation, in particular whey or asolution of whey proteins.

More in particular, this embodiment relates to a method for preparing aprotein preparation having a reduced casein content and comprising atleast one protein constituent other than casein/caseinate, starting froma protein preparation which comprises the at least one proteinconstituent and casein/caseinate, in which the starting preparation isbrought into contact with a proteolytic enzyme under conditions for thehydrolysis of the casein/caseinate, characterized in that thecasein/caseinate and preferably also the at least one proteinconstituent are in an essentially dissolved state, and in that aproteolytic enzyme is a proteinase specific for casein/caseinate, suchthat the casein/caseinate is hydrolysed while the at least one proteinconstituent remains essentially intact.

In this embodiment of the invention, a casein-specific proteinase asdescribed above is likewise used, in particular for the specificdegradation of the casein/caseinate in the presence of the at least onefurther protein constituent.

Other than in the above-described embodiment, however, subsequently theone or more further (milk) protein constituents which have essentiallyremained intact are obtained, instead of the casein hydrolysisfragments.

A casein epitope is to be understood herein as those parts of the caseinmolecules or casein fragments in the starting material which react withan antibody to casein.

A protein preparation free from casein epitopes is to be understoodhereinafter as a protein preparation which does not contain any proteinsor protein fragments which are able to form a bond with antibodies to acasein epitope (cross-reaction).

Such preparations stripped of casein, casein fragments and/or caseinepitopes are important because of their reduced immunologicalcharacteristics. Thus, Cavallo et al., The Lancet, 1996; 348:926-928report that β-casein is possibly involved in insulin-dependant diabetes,since early consumption of cow's milk may cause a specific immuneresponse to β-casein, which cellular and humoral anti-β-casein immuneresponse may produce a cross-reaction with proteins on the pancreaticbeta cell, inter alia because of the high homology between the sequencesof β-casein and various beta cell molecules. In this respect, milkprotein preparations stripped of casein may play an important part inthe preparation of non-immunogenic infant formulas.

The present embodiment of the invention generally comprises thefollowing steps:

a. bringing the protein preparation, which comprises the at least onedesired protein constituent and casein/caseinate, into contact with aproteinase specific for casein/caseinate, such that the casein/caseinateis hydrolysed while the desired protein constituent remains essentiallyintact,

b. separating the casein hydrolysis fragments from the desired proteinconstituent which has essentially remained intact.

This embodiment of the invention can be used for the selective removalof casein/caseinate from casein-containing protein preparations, toprovide a protein preparation stripped of casein, the selective removalof even low to very low casein levels being possible in the process.

This embodiment can be used, in particular, to remove casein from milkor a solution of milk proteins, and in particular from whey or asolution of whey proteins.

The casein level of the starting preparation will, as a rule, be atleast 0.1 wt %, in particular 1 wt % or more, although even loweramounts of casein can also be selectively removed. The upper limit forthe casein content is not essential, although in practice the caseincontent in the starting material may generally be less than 50% byweight, preferably less than 20% by weight, more preferably less than10% by weight.

The hydrolysis step is essentially performed as described above forembodiment a, using the same casein-specific proteinases. Anyadaptations required, depending on the starting preparation used, willbe evident to those skilled in the art, also drawing on the presentdescription.

The degree of hydrolysis will preferably be such that after completionof the hydrolysis step, more than 90%, more preferably to more than 99%,and most preferably essentially all casein present in the startingpreparation has been cleaved into smaller fragments. The level of stillintact casein molecules after the hydrolysis step is preferably lessthan 0.1 wt % or more preferably less than 0.01 wt %.

The hydrolysis is preferably carried out to such a degree that the levelof antigenic casein hydrolysis fragments (including any uncleaved/intactcasein) is less than 1 wt %, preferably less than 0.1 wt %.

According to a particular embodiment, the protein preparation obtainedafter hydrolysis and containing the casein hydrolysis fragments, isalready free from casein epitopes, which means that the casein and/orthe (antigenic) fragments thereof have been cleaved to such a degreethat casein epitopes can no longer be detected in suitable immunologicalassays, such as ELISA, using casein-specific antibodies.

However, as a rule, such extensive hydrolysis is not required to obtaina protein preparation free from casein epitopes, since any smallantigenic casein hydrolysis fragments still present can likewise beremoved in the course of a subsequent separation step, as describedbelow.

It will also be clear to those skilled in the art that this embodimentcan be used not only to remove/separate off intact casein molecules butalso to remove/separate off casein fragments, in particular antigeniccasein fragments.

To separate off the casein fragments obtained by hydrolysis, anysuitable separation method can be used, a separation method on the basisof molecular mass and/or molecular size again being preferred. Inparticular, ultrafiltration or a similar technique can be used, thedesired fraction not being the permeate (as in embodiment a. above) butthe retentate which will contain the one or more desired proteinconstituents.

In ultrafiltration, a membrane having a suitable molecular cut-off valueis used. Dependent upon said cut-off value, solely the casein hydrolysisfragments can be separated off, or—by using a suitably higher cut-offvalue—simultaneously one or more of the small(er) further proteinconstituents, which have remained intact, can also be removed.

The removal of essentially only the casein hydrolysis fragments willgenerally involve the use of a membrane having a molecular cut-off valueof 10 kDa or less, depending on the degree of hydrolysis.

The degree of hydrolysis and the molecular cut-off value of theultrafiltration membrane used are preferably chosen such that the levelof (antigenic) casein hydrolysis fragments and/or casein epitopes in theretentate obtained after ultrafiltration is less than 0.1 wt %,preferably less than 0.01 wt%.

Most preferably, the degree of hydrolysis and the molecular cut-offvalue are chosen such that all antigenic casein hydrolysis fragmentsand/or casein epitopes can be separated off, so that it is no longerpossible to detect, by means of suitable immunological assays usingcasein-specific antibodies, such as ELISA, any epitopes of casein in theprotein preparation obtained after ultrafiltration. Such a preparationis here referred to as “free from casein epitopes”.

If the starting material is milk, whey or a solution of milk proteins orwhey proteins, what is obtained after the casein hydrolysis fragmentshave been separated off will generally be a mixture of proteins. Thismixture may be further separated according to methods known per se, toobtain the separate protein constituents in purified or essentially pureform. This can be done, for example, by precipitation, chromatographictechniques, or by further separation on the basis of molecular mass ormolecular size. It is thus possible, by successive ultrafiltration stepswith increasing molecular cut-off values of the membrane, to achievefractionation of the milk proteins.

This embodiment of the invention can be used, in particular, to obtainmilk protein preparations, in particular preparations comprising one ormore whey proteins such as α-Lac, β-Lg, BSA and IgG. However, thisembodiment can also be used for purifying or isolating proteins notnative to natural milk, starting from a casein-containing medium. Allthese protein preparations are preferably free from casein epitopes, asdefined above.

The (whey) protein preparations thus obtained, in particular thepreparations free from casein epitopes, can be used for preparing foodpreparations such as infant formula, for preventing immunologicalreactions against casein, in particular β-casein.

With respect to the invention it is further noted that it differs fromthe action of bacterial proteinases during the maturation ofcheese—which is outside the scope of the invention—in that thecasein/caseinate, and preferably also the further protein constituents,during the hydrolysis are in solution, which may or may not beconcentrated and will generally be aqueous; in cheese-making, thecasein-cleaving enzymes act on the precipitated casein in the curd.

Moreover, the cleavage of casein in the cheese milk takes place underthe influence of a specific enzyme (a curdling enzyme such as chymosin).The action, and particularly the specificity, of the proteinases usedaccording to the invention differ demonstrably, however, from that ofsuch curdling enzymes. Thus, according to one aspect of the invention,the specific action of chymosin on casein, in particular with respect tothe cleavage of the phenylalanine/methionine bond, is disclaimed, eventhough the invention in the widest sense is not limited to a specifictype of enzymatic reaction.

Although the invention has been described above with respect to twodifferent embodiments, it should be understood that these embodimentscan also be combined in that, after the specific hydrolysis of thecasein/caseinate-containing (milk) protein preparation, both the caseinhydrolysis fragments and the proteins which have remained intact areobtained as two separate fractions by means of a suitable separationtechnique as described above. These fractions can then be furtherprocessed and/or used in the above-described manner. Other adaptationsand uses of the teachings of the invention, which are within the scopeof the following claims, will be obvious to those skilled in the art.

The invention will be explained below with reference to the present,nonlimiting example and the appended figures showing the following:

FIG. 1: Degradation of the various milk proteins during incubation withproteinase Wg2 (15 units/mg protein) expressed as percentage intactprotein. (+) BSA; (Δ) BSA (90 units/mg protein); (◯) βLg; (▪) IgG; (▴)αLa; () total casein; (∇) total casein (90 units/mg protein).

FIG. 2: RP-HPLC analysis of hydrolysates in which a mixture of totalcasein and BSA was hydrolyzed with proteinase Wg2 (90 units/mg protein).Intact BSA and casein fragments were subsequently separated by means ofultrafiltration (cut-off value 30 kDa ); (A) Mixture of casein and BSA,t=0 hours; (B) Mixture of casein and BSA, t=22 hours; (C) 30 kDapermeate fraction of the t=22 hours hydrolysate; (D) 30 kDa retentatefraction of the t=22 hours hydrolysate.

FIG. 3: ABBOS residual antigenicity of retentate and permeate fractionsobtained after ultrafiltration (cut-off values 10 and 30 kDa ) of amixture of total casein and BSA hydrolyzed with proteinase Wg2 (90units/mg protein); (▪) synthesized ABBOS-peptide; (Δ) 10 kDa retentate;(◯) 30 kDa retentate; (+) 10 kDa permeate; (▴) 30 kDa permeate.

FIG. 4: Specific IgE binding to casein and the whey proteins of serafrom patients with cow milk allergy and persons not allergic to milkproteins.

FIG. 5: IgE residual antigenicity of retentate and permeate fractionsobtained after ultrafiltration (cut-off value 3 and 5 kDa ) of a mixtureof casein, α-lactalbumin, β-lactoglobuline or of sodium caseinatehydrolyzed with proteinase Wg2 (90 units/mg protein); (◯) intact milkprotein: upper row from left to right αLa, βLg and BSA, lower row fromleft to right IgG and casein; (⋄) 5 kDa retentate of mixture casein, αLaand βLg; () 5 kDa retentate of sodium caseinate; (Δ) 5 kDa permeate ofmixture casein, αLa and βLg; (t) 5 kDa permeate of sodium caseinate; (+)3 kDa permeate of mixture casein, αLa and βLg.

FIG. 6: Schematic representation of embodiments “A” and “B” of theinvention.

EXAMPLE

a. Preparation and Purification of the Proteinase

The organism Lactococcus lactis subsp. cremoris strain WG2 (collectionNetherlands Institute for Dairy Research B1021) was grown (overnight at30° C.; final pH 4.6-4.8) in milk and stationary-phase cells weresubsequently harvested as described by Exterkate in Appl. Environ.Microbiol. 47:177-183 (1990). Essentially according to Mills and Thomasin N. Z. J. Dairy Sci. Technol. 13:209-215 (1978) the lactococcal cellenvelope proteinase Wg2 (proteinase Wg2) was autoproteolyticallyreleased by incubating the cells in a calcium-free buffer at 30° C. (20mM imidazole-HCl buffer pH 6.4). After incubating, the cells weresedimented by centrifugation (15 min at 15,000 g). Supernatant fractionswere pooled, extensively washed with the same buffer supplemented with10 mM CaCl₂ and concentrated. For further purification the crudeproteinase Wg2 concentrate was centrifuged (30 min at 48,000 g),filtered (0.45 μm) and applied onto a MonoQ HR 10/10 (Pharmacia,Uppsala, Sweden) anion-exchange column, equilibrated with 20 mMimidazole-HCl buffer (pH 6.4) supplemented with 10 mM CaCl₂. Afterelution of non-binding material, proteins were eluted at 2 ml/min with a0-0.3 M NaCl gradient in the same buffer. Fractions containing theenzyme activity, and without activity of the intracellular exopeptidasePepN, were pooled, concentrated, aliquotted and stored at −20° C. Beforeuse, the enzyme activity of the lactococcal proteinase Wg2 wasquantified and contamination with the intracellular exopeptidase PepNwas tested using two different chromogenic substrates: S-2586 andLys-pNA, respectively as described by Exterkate et al in Appl.Microbiol.Biotechnol. 33:401-403 (1990). One proteinase unit representsthe formation of 1 nmol p-nitroanilide per minute at 30° C.

b. Hydrolysis Experiments

Two different series of hydrolysis experiments were performed. In thefirst series the various milk proteins, total casein, αLa, βLg, BSA orIgG were separately dissolved in 50 mM imidazole-HCl buffer (pH 6.5) forthe incubation with the proteinase Wg2. The final protein concentrationwas 10 mg/ml, the enzyme activity 15 or 90 units/mg protein and thetemperature 30° C. Aliquots of 20 μl were taken out of the incubationvessels at 0, 1, 3, 5 and 22 hours after the addition of lactococcalproteinase Wg2.

In the second series of hydrolysis experiments commercially availablesodium caseinate and a mixture of equal amounts of total casein, αLa andβLg (on a protein basis) were dissolved in 50 mM imidazole-HCl buffer(pH 6.5) and offered as a substrate for the proteinase Wg2. The totalprotein concentration was 6 mg/ml and the enzyme activity 90 units/mgprotein. After hydrolysis for 22 hours at 30° C., the hydrolysates wereultrafiltered, using membranes (Omega-type, Filtron, Northborough,Mass., USA) with cutoff values ranging from 3 to 30 kDa .

Because BSA became insoluble in the imidazole-HCl buffer after heating,necessary to expose the ABBOS epitope for immunological detection (seebelow), a mixture of equal amounts of total casein and BSA was dissolvedin distilled water and hydrolyzed using the pH-stat technique asdescribed by Adler -Nissen J. Chem. Tech Biotech. 32:138-156 (1982).During the hydrolysis with proteinase Wg2 the pH was kept constant at pH7.0 by the addition of 0.05 M NaOH. The total protein concentration was4 mg/ml and the enzyme activity 90 units/mg protein. After hydrolysisfor 22 hours at 30° C., the hydrolysates were ultrafiltered, usingmembranes (Omega-type, Filtron, Northborough, Mass., USA) with cut-offvalues ranging from 3 to 30 kDa.

c. Physicochemical and Immunological Characterization of theHydrolysates

To follow the degradation of the milk proteins during hydrolysis RP-HPLCas described by Visser et al. in J. Chromatogr.548:361-370 (1991) wasapplied. The decrease of the intact-protein peak areas was quantitatedby integration (Turbochrom, Perkin Elmer Co., Cupertino, Calif., USA).The occurrence of cross-reactivity between the ABBOS peptide and thevarious proteinase Wg2 hydrolysates was determined by competitiveenzyme-linked immunosorbent assay (ELISA), using rabbit anti-ABBOS serumas described by van Beresteijn and Meijer in Diabetes Care 19:1364-1369(1996). In order to expose the ABBOS epitope, hydrolysates were heatedfor 5 min at 95° C. The same method was applied to determine thepresence of cross-reactive milk-protcin-specific IgE antibodybindingsites in the various proteinase Wg2 hydrolysates as described by vanBeresteijn et al. in J. Allergy Clin Immunol. 96:365-374 (1995) using ahuman antiserum from cow milk alllergic patients with high IgE antibodytiters to all individual milk proteins (FIG. 4).

d. Results

Enzyme Specificity

The specificity of the lactococcal proteinase Wg2 for the various milkproteins is shown in FIG. 1. Degradation of protein under the chosenconditions was only detectable when total casein was used as asubstrate; the whey proteins αLa, βLg, BSA and IgG remained intact. Theapparent increase in RP-HPLC peak area for IgG (FIG. 1) cannot beexplained, but may be connected with the observed insolubilization ofIgG after thawing of the hydrolysate samples. Increasing the enzymeconcentration to 90 units/mg protein total casein was degraded within 5hours (FIG. 1).

Separation of Casein and Whey Protein(s)

The discriminating behaviour of proteinase Wg2 leading to the specificdegradation of casein in the presence of the whey proteins (αLa, βLg,BSA and IgG) caused an increased difference in molecular weight betweenthe smaller casein fragments and the still-intact whey proteins.Dependent on the cut-off value of the membrane, specific removal ofintact whey proteins from the casein fragments was possible. Membraneswith cut-off values up to 30 kDa selectively removed BSA from a mixtureof total casein and BSA or from a commercially available sodiumcaseinate (generally contaminated with whey proteins) after treatmentwith proteinase Wg2. FIG. 2 shows the specific degradation of totalcasein by proteinase Wg2 in the presence of BSA with subsequent removalof the intact BSA using a 30 kDa membrane.

Residual ABBOS Antigenicity

RP-HPLC analysis could not detect BSA or fragments thereof in permeatefractions obtained after hydrolysis of a mixture of total casein and BSAor sodium caseinate with proteinase Wg2 followed by ultrafiltration(FIG. 2). Using the more sensitive immunological method these resultswere confirmed. FIG. 3 shows that the rabbit anti-ABBOS serum did notshow any binding inhibition with the permeate fractions up to aninhibitor concentration of 1 g/l. In contrast, the retentate fractionsand the control (synthesized ABBOS peptide) showed 100% inhibition atthat protein concentration.

Residual IgE Antigenicity

Treatment of a mixture of total casein, αLa and βLg or of sodiumcaseinate with proteinase Wg2 followed by ultrafiltration throughmembranes with lower cut-off values (5 kDa or 3 kDa ) addditionallyremoved the lower molecular weight antigenic whey proteins. FIG. 5 showstypical examples of the inhibition ELISA for βLg, αLa, BSA, IgG andtotal casein for these hydrolysates. The human antiserum did not showany IgE binding inhibition with the permeate fractions up to aninhibitor concentration of 1 g/l. In contrast, the retentate fractionsand the respective intact milk protein showed 100% inhibition at thatprotein concentration.

What is claimed is:
 1. A method for producing a casein/caseinatehydrolysate that is stripped of immunogenic protein other thancasein/caseinate, the method comprising the steps of: a) providing anaqueous solution containing essentially dissolved casein/caseinate andan essentially dissolved immunogenic protein other thancasein/caseinate; b) adding to the aqueous solution a casein-specificproteolytic enzyme; c) incubating the aqueous solution and thecasein-specific proteolytic enzyme under conditions such that thecasein/caseinate is hydrolysed while the immunogenic protein other thancasein/caseinate remains essentially intact; d) separating thehydrolysed casein/caseinate and the essentially intact immunogenicprotein other than casein/caseinate; and e) recovering the hydrolyzedcasein/caseinate stripped of immunogenic protein.
 2. The method of claim1, wherein the immunogenic protein other than casein/caseinate is a wheyprotein.
 3. The method of claim 2, wherein the whey protein is bovineserum albumin.
 4. The method of claim 3, wherein essentially nodetectable antigenic components of the ABBOS peptide are present in thecasein/caseinate hydrolysate.
 5. The method of claim 4, whereinessentially no detectable antigenic components of bovine serum albuminare present in the casein/caseinate hydrolysate.
 6. The method of claim1, wherein the aqueous solution is selected from the group consisting ofmilk, a fraction of milk, and an aqueous solution of milk proteins. 7.The method of claim 1 wherein the casein-specific proteolytic enzyme isselected from the group consisting of a Lactococcus cell wallproteinase, a collagenase and a dispase.
 8. The method of claim 1,wherein the casein-specific enzyme is selected from the group consistingof a cell wall proteinase of Lactococcus lactis subsp. cremoris strainWg2, a Clostridium histolyticum collagenase and a Bacillus polymyxadispase.
 9. The method of claim 1, wherein the separation in step (d)uses a separation method based on a characteristic selected from thegroup consisting of molecular mass and molecular size.
 10. The method ofclaim 9, wherein the separation method is ultrafiltration.
 11. Themethod of claim 10, wherein the ultrafiltration uses a molecular cut-offvalue in the range of 10-30 kDa.